Drosophila melanogaster was used to identify genes with a potential role in genetic regulation of purine biosynthesis. In this study we examine two dominant genetic modifiers of the essential gene Prat, which encodes amidophosphoribosyltransferase (EC 2.4.2.14). We found that Mod(Prat:bw)3-1 enhances Prat expression only in female heads, whereas Mod(Prat:bw)3-5 suppresses Prat in all stages and tissues examined for both sexes. For Mod-3-5, gene expression microarrays were used to identify other genes that are affected by the modifier. Three mapping approaches were used to localize these modifiers. Deficiency and meiotic mapping showed that the complex lethal complementation group previously associated with Mod-3-1 and Mod-3-5 is actually due to shared second-site lethal mutations. Using male recombination mapping, Mod-3-1 was localized to a 21 kilobase region containing nine genes, and Mod-3-5 was localized to a 53 kilobase region containing eight genes.